In mammals, there are three chemosensory systems (taste, olfactory and vomeronasal perceptions) that function to convert external chemical signals to specific neuronal activities. These neuronal signals are then integrated in different regions of brain and the output of these signals affect the organism's various innate behaviors, ranging from aversion and attraction to food or small volatile chemicals to reproductive actions. Among these chemosensory systems, taste perception provides immediate valuation of nutrients. Although the molecular universe of tastants consists of diverse chemical structures such as ions, small organic molecules, proteins, carbohydrates, amino acids, and lipids, it is generally believed that mammals have five basic taste modalities: sour, salty, bitter, sweet, and umami (glutamate) as described, e.g., in Lindemann, Physiol. Rev. 76:718-766, 1996; Kinnamon et al., Annu. Rev. Physiol. 54:715-731, 1992; and Gilbertson et al., Curr. Opin. Neurobiol. 10: 519-527, 2000.
The sensation of taste is initiated by the interaction of tastants with their receptors in the taste cells, which are clustered in onion-shape taste buds embedded within the lingual epithelium in tongue and palate as described, e.g., in Lindemann, supra. On the tongue, taste buds are topographically distributed into papillae in different locations of tongue. Fungiform papillae are located at the front of the tongue and contain a small number of taste buds; foliate papillae, containing dozens of taste buds, are localized along the posterior lateral edge of the tongue; and at the back of the tongue, circumvallate papillae contain thousands of taste buds. Classical physiological studies have found that fungiform papillae are sensitive to sweet, foliate papillae are sensitive to sour and bitter, and circumvallate papillae are particularly sensitive to bitter.
Each taste modality is thought to be mediated by distinct cell surface receptors that are expressed in a subset of taste cells. Electrophysiological and biochemical studies suggest that salty and sour tastants signal through Na+ and H+ membrane channels as described, e.g., in Heck et al. Science 223: 403-405, 1984; Avenet et al., J. Memb. Biol. 105:245-255, 1988, Doolin et al., J. Gen. Physiol. 107:545-554, 1996; Formaker et al., Am. J. Physiol 255:1002-1007, 1988; Kinnamon et al. Proc. Natl. Acad. Sci. USA 85:7023-7027, 1988; and Gilbertson et al., J. Gen. Physiol. 100:803-824, 1992. In contrast, bitter, sweet, and umami taste transduction are believed to involve G protein-coupled receptors (GPCR).
GPCRs are a class of seven-transmembrane proteins which transduce an extracellular signal, i.e., ligand binding to receptor, into a cellular response. Upon ligand binding to a GPCR, the GPCR activates an intracellular guanine nucleotide protein known as G-protein (guanine nucleotide binding protein), which mediates a response to the extracellular signal. G-proteins are heterotrimeric proteins composed of an alpha, beta and gamma subunit. The activated G protein alters the activity of various cellular effector enzymes (e.g., adenylate cyclase and phosphodiesterase), which in turn alters the levels of various second messengers (e.g., cAMP, cGMP, and inositol triphosphate (IP3)).
Experiments with the bitter substance, denatonium, have shown that the secondary messages, cAMP and IP3, are induced in response to bitter stimuli as described, e.g., in Spielamn et al., Am. J. Physiol. 270:C926-C931, 1996; and Ruiz-Avila et al., Nature 376:80-85, 1995. Other studies have revealed that gustducin, a G protein expressed in subpopulation of taste buds, can activate phosphodiesterase (PDE) and thereby decrease cNMP levels in response to bitter stimuli as described, e.g., in Ruiz-Avila et al, supra; and Hoon et al., Biochem. J. 309:629-636, 1995. These secondary messages, which are generally involved in G protein signaling, are consistent with the involvement of GPCRs in taste transduction. Sweet substances have also been shown to cause the elevation of the secondary messages, cAMP and IP3, presumably in response to activation of G protein-coupled receptor cascades by Gs protein as described, e.g., in Striem et al., Biochem. J. 260:121-126, 1989; and Bernhardt et al., J. Physiol. 490:325-336, 1996. The involvement of G proteins in bitter and sweet transduction is also supported by the discovery that mice with a null allele of gustducin have an impaired ability to detect bitter and sweet substances as described, e.g., in Wong et al., Nature 381:796-800, 1996.
The involvement of G-protein coupled receptors in taste transduction has recently been confirmed by the discovery of three families of GPCRs expressed in mammalian taste bud cells, a number of which have been shown to be activated by bitter and glutamate tastants as described, e.g., in Firestein, Nature 404:552-553, 2000. A splice variant of a metabotropic glutamate receptor was cloned from rat taste bud and was shown to respond to monosodium L-glutamate when expressed in heterogonous cells as described, e.g., in Chaudharri et al., Nature Neurosci. 3:113-119, 2000. Two additional candidate taste receptors, T1R1 and T2R2, have been isolated from rat taste bud, and show distant homology with putative pheromone receptor V2Rs and metabotropic glutamate receptors, as described in Hoon et al., Cell 96:541-552, 1999. T1R1 and T2R2 were postulated to function as sweet and bitter receptors, respectively, based on their topographic distribution in the tongue as described, e.g., in Hoon et al., supra, 1999. Searches of the human and mouse genomes have identified another family of taste receptors (T2Rs) containing approximately 25 members as described, e.g., in Adler et al., Cell 100:693-702, 2000; and Matsunami et al., Nature 404:601-603, 2000. One receptor in this family, mT2R5, is specifically activated by the bitter substance cycloheximide, while the human hT2R4 and mouse mT2R8 respond to denatonium as described, e.g., in Chandrashekar et al., Cell 100:703-711, 2000.
Over the past few years, much effort has been directed toward the development of various sweeteners that interact with taste receptors to mimic natural sweet taste stimulants. See, Robert H. Cagan, Ed., Neural Mechanisms in Taste, Chapter 4, CRC Press, Inc., Boca Raton, Fla., 1989. Examples of sweeteners that have been developed to mimic sweet tastes are saccharin (an anhydride of o-sulfimide benzoic acid), monellin (a protein), aspartame (a peptide composed of aspartic acid and methyl ester of phenylalanine) and the thaumatins (also proteins). Many sweeteners developed to date are not suitable as food additives, however, because they are uneconomical, high in calories, carcinogenic or lose their sweetness when exposed to elevated temperatures for long periods, rendering them unsuitable for use in most baking applications.
Development of new sweeteners that mimic sweet (and other) tastes has been limited by a lack of knowledge of the taste cell proteins responsible for transducing the sweet taste modalities. Accordingly, the identification of new sweet taste receptors would enable the identification of the natural ligands, i.e., natural sweet tastants, of these proteins and the design of novel sweeteners that mimic sweet taste perception. The present invention fulfills these and other needs.